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PubMed1979

A sensitive microassay for the murine alternative complement pathway.

A microhemolytic assay for measurement of murine alternative complement pathway activity is described. The assay uses 51Cr release from neuraminidase-treated rabbit erythrocytes incubated with Mg2+ EGTA-chelated murine serum. Neuraminidase pretreatment of rabbit erythrocytes increases the sensitivity of the assay 8--10-fold, enable the use of small volumes of individual mouse sera. The assay affords a simple, sensitive and reproducible method for measuring murine alternative complement pathway activity. Significant differences were found between strains alternative complement pathway activity of serum from various inbred murine lines was measured.

Animals↗
PubMed1979

Consumption of C3 via the classical and alternative complement pathways by sera and synovial fluids from patients with rheumatoid arthritis.

Five sera and four synovial fluids from patients with rheumatoid arthritis were found to contain substances which consumed hemolytic C3 in normal human serum (NHS) and in normal guinea pig serum (NGPS). These fluids were then tested for ability to activate the alternative pathway by incubating them with NHS containing MgEGTA and C4 deficient GPS. All sera and two synovial fluids depleted C3 in these reagents, indicating direct activation by the alternative pathway. Density gradient ultracentrifugation demonstrated C3 fixing activity in some specimens in the greater than 19s regions. These substances may also activate complement similarly in vivo and participate in the development of inflammatory processes associated with this disease.

Adult↗
PubMed1979

[Alternative complement pathway (author's transl)].

The dual role of the alternative complement pathway in recognition of foreign substances by a non-immune host and in the intrinsic regulation of the complement sequence is now well recognized. Activation of this pathway occurs through escape from its regulatory mechanisms induced by the activating principle; its functional expression depends on the respective levels of the component proteins C3, factor B, factor D and properdin, and on the control proteins beta 1H and C3bINA. This article presents recently acquired knowlege on the molecular mechanisms of activation, regulation and behaviour under pathological conditions of the alternative complement pathway.

Complement Activation↗
PubMed1979

Alternate complement pathway activation by group A streptococci: role of M-protein.

Avirulent strains of group A streptococci readily activate the complement system in normal human serum via the alternate complement pathway (ACP). Virulent M-positive group A streptococci are much less potent as activators of the ACP. The ability of M-positive streptococci to activate the ACP is enhanced by trypsinization or mild peptic digestion. The latter treatment removes the serologically active and antiphagocytic type-specific moieties of M protein, but retains the surface fuzzy layer. The phagocytosis of avirulent streptococci is markedly enhanced by preopsonization in serum chelated with Mg-ethylene glycol tetraacetic acid (classic complement pathway blocked) but not in serum devoid of heat-labile factors. These studies suggest that the function of M protein as a virulence factor may be mediated, at least in part, by its ability to retard interaction of ACP components with structures present on the streptococcal cell surface.

Bacterial Proteins↗
PubMed1990

Complement alternative pathway activation and control on membranes of human lymphoid B cell lines.

Membrane regulatory molecules normally prevent complement activation by autologous cells, therefore we compared the membrane control system of human lymphoid cell lines which activate or not human complement through the alternative pathway (AP). Membrane expression of decay-accelerating factor (DAF), membrane cofactor protein (MCP), complement receptors (CR)1, CR2 and H was measured either by radioimmunoassay or enzyme-linked immunosorbent assay on cell lysates. Soluble extracts of isolated membranes were tested functionally for their ability to accelerate the decay of C3bBb C3-convertase and allow the cleavage of C3b by factor I. Both regulatory functions were detected in solubilized membranes of Ramos cells, which do not activate the AP, as well as on the potent AP activator, Raji. Raji cells were found to express CR2, DAF and MCP molecules, while MCP was the only known regulatory protein detected on Ramos cells which expressed neither CR1, nor CR2, H or DAF. The I-cofactor activity of both Raji and Ramos cells was immunoprecipitated by anti-MCP, but the decay-accelerating activity was not adsorbed by anti-DAF nor by any of the available antibodies. Two EBV genome-negative cell lines (BJAB, BL41) were tested before and after in vitro conversion by EBV. As previously shown, EBV-converted cell lines activate the AP more efficiently than EBV- cell lines. At the same time, EBV superinfection induces an increase of both AP regulatory functions of cell membranes and enhances the expression of DAF, MCP and CR2. The results of this study show that complement activation by lymphoid cell lines is not related to an impaired autologous control of these cells, but that the expression of regulatory molecules increases together with the appearance of activating structures on the cell surface. Our results also suggest the occurrence of a new factor involved in the decay-accelerating activity on BL lines.

Antigens, CD↗
PubMed1978

Antibody-dependent cytotoxicity against Trypanosoma rhodesiense mediated through an alternative complement pathway.

A quantitative in vitro method was used to examine the role of classical and alternative pathways of complement activation in cytotoxicity against African trypanosomes by immune serum. This assay is based on the estimation of the extent of antibody-mediated cytotoxicity by measurement of inhibition of incorporation of [(3)H]leucine as an indicator of trypanosome metabolic integrity. To determine which pathway(s) is activated during cytotoxic events, complements sufficient in all components (C4S) and deficient in C4 (C4D) were used. Immune inhibition of [(3)H]leucine uptake by trypanosomes was observed in the presence of both complement sources. Treatment of C4S or C4D serum with cobra venom factor or disodium ethylenediaminetetraacetic acid abolished antibody-mediated cytotoxicity as well as immune hemolysis, thus suggesting the requirement for late-acting complement components. Ethyleneglycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid had no effect on inhibition of leucine incorporation, whereas immune hemolysis was inhibited, suggesting that cytotoxicity did not require C1 activation, a Ca(2+)-dependent event. The dependence of the cytotoxic process on Mg(2+) and not Ca(2+) ions and the fact that C4D guinea pig serum is fully active in trypanosome cytotoxicity indicate that an alternative pathway of complement activation is sufficient for activity.

Animals↗
PubMed1978

Participation of immunoglobulin and the alternative complement pathway in opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron.

Studies were conducted to determine the requirements for immunoglobulin and complement for opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron. The ability of human sera depleted of immunoglobulin or complement components to promote phagocytosis and intracellular killing of the strains of Bacteroides by human leukocytes was measured in vitro under anaerobic conditions. Neither hypogammaglobulinemic sera nor pooled normal human serum (PNHS) heated at 56 C for 30 min supported phagocytosis and killing of the strains of Bacteroides. Sera depleted of terminal complement components by treatment with inulin or cobra venom factor and C8-deficient human serum did not support phagocytosis of the test strains. PNHS depleted of C3, factor B, or factor D also did not support phagocytosis of either strain. Dose-dependent restoration of the opsonic activity of factor B-depleted serum was accomplished by purified human factor B but not by human C2. The results indicated that immunoglobulin and components of the alternative complement pathway participate in opsonization of the strains of Bacteroides tested in this study.

Agammaglobulinemia↗
PubMed1978

The importance of C5 and the role of the alternative complement pathway in leukocyte chemotaxis induced in vivo and in vitro by Bacteroides fragilis lipopolysaccharide.

Chambers implanted subcutaneously in C5 normal (C5 N) and C5 deficient (C5 D) mice were used to examine the migration of polymorphonuclear leukocytes (PMNs) into the wound chamber fluid in response to injected Bacteroides fragilis lipopolysaccharide (LPS). The difference in PMN migration was highly significant between the two mouse strains, the C5 D mice showing no initial, but a low, delayed migration. The results from the study indicated that chemotaxis plays a major role in the accumulation of PMNs in the acute inflammatory response. Intraperitoneal endotoxin stimulation also showed a significantly lower total number of leukocytes in the exudate from C5 D mice as well as a delayed migration of cells compared to C5 N mice. No leukotactic mediators were elaborated in C5 D serum or exudate upon incubation with LPS when tested in a modified Boyden chamber. However, endotoxin-induced wound chamber fluid in C5 D mice showed an increasing leukotactic activity at the same time as the acute inflammatory response subsided in C5 N mice. Incubation of B. fragilis LPS in C4 deficient (C4 D) guinea pig serum indicated that the LPS was able to activate complement components to generated split products chemotacic for rabbit PMNs via the alternative complement pathway.

Animals↗
PubMed1979

Lysis of measles virus-infected cells by the purified cytolytic alternative complement pathway and antibody.

The dependence of antibody-and-complement-mediated lysis of virus-infected cells on the alternative pathway was examined utilizing the isolated cytolytic alternative pathway--a system consisting of the six purified proteins of the alternative pathway of activation (C3, factors B and D, beta 1H, C3b inactivator and properdin), and the five proteins of the membrane attack pathway (C5--9) of complement. HeLa cells acutely infected with measles virus were lysed by anti-viral IgG and the isolated cytolytic alternative pathway with an efficiency comparable to whole human serum. IgG and its F(ab')2 fragment were equally effective in inducing lysis by the isolated cytolytic alternative pathway, binding of approximately equal to 5 X 10(7) molecules per cell being required for 50% lysis; in contrast, no lysis occurred when equivalen or greater amounts of Fab' were bound to the virus-infected cell. Properdin was required for lysis. No lysis occurred if properdin was deleted from the isolated cytolytic alternative pathway, and lysis was diminished by 80% in properdin-depleted serum. Uptake of [125I]C3b from the isolated alternative pathway onto measles virus-infected cells occurred in the absence of properdin, but was accelerated in the presence of properdin. The 11 proteins of the isolated cytolytic alternative pathway are thus sufficient for lysis of measles virus-infected cells bearing anti-viral IgG or F(ab')2 without any other serum protein.

Antibodies, Viral↗
PubMed1979

Inhibition of alternative complement pathway opsonization by group A streptococcal M protein.

Group A streptococcal M protein is known to be antiphagocytic; however, the exact basis for this property has not been established. In this study the hypothesis was tested that cell wall--associated M protein inhibits phagocytosis by interfering with bacterial opsonization. Two strains of group A Streptococcus pyogenes, CS44 (M+) and CS64 (an M- variant of CS44), were radiolabeled, and after incubation in serum these organisms were exposed to human polymorphonuclear leukocytes. Phagocytosis was quantitated by measurement of leukocyte-associated radioactivity. The contributions of complement and of immunoglobulin to streptococcal opsonization were evaluated by use of serum from a variety of sources. The results revealed that the M- strain was efficiently opsonized via the alternative complement pathway in a relative absence of immunoglobulins. In contrast, the M+ strain was poorly opsonized by all sera tested. These findings suggest that streptococcal M protein in some way prevents bacterial opsonization via the alternative complement pathway and that this property of M protein may partly explain its antiphagocytic characteristic.

Antigens, Bacterial↗
PubMed1991

Specific antibodies enhance alternative complement pathway activation by cuprophane.

Cuprophane activates the human alternative complement pathway. We have observed that the extent to which the alternative pathway was activated in the presence of fixed amounts of cuprophane in vitro greatly differed between individuals. Several lines of evidence indicated a role for anti-dextran antibodies in determining the extent of alternative pathway activation by cuprophane in whole serum: (1) alternative pathway activation by cuprophane was directly related to the extent to which it was activated by Sephadex in a given serum; alternative pathway activation by Sephadex was previously shown to be antibody-dependent; (2) alternative pathway activation by cuprophane was related to the serum titre of anti-dextran IgG antibodies; (3) adsorption of serum with Sephadex or cuprophane reduced the ability of the serum to be activated in the presence of fresh cuprophane. These results suggest that measuring serum values of anti-dextran antibodies may be potentially helpful for evaluating patients at increased risk of complement activation during haemodialysis with cuprophane membranes.

Antibodies↗
PubMed1991

Simple quantitative haemolytic microassay for determination of complement alternative pathway activation (AP50).

We describe here a simple, reliable and quantitative method to measure the alternative pathway (AP) dependent mode of complement activation for the lysis of rabbit erythrocytes. In the test the reciprocal of the plasma volume needed to destroy 50% of available rabbit erythrocytes is defined as the functional measure of this activity (AP50). The test was found to be highly reproducible both within and between assays with a coefficient of variation which was less than 5%. Sensitivity was also shown to be satisfactory and all of the plasma samples from the healthy blood donors which were tested could be assayed with precision. The specificity of the AP50 assay for AP complement activation was verified by the fact that a C4 affinity-depleted plasma gave an AP50 value within the normal range (52.8 U/ml) while a similar aliquot of the same plasma, affinity-depleted of factor B, gave an undetectable AP50 value (less than 10 U/ml). Furthermore, a sample was unable to lyse the target cells when heated to 50 degrees C or 56 degrees C, treatments which are known to destroy factor B and total haemolytic complement, respectively. To ensure inhibition of the classical pathway of activation. EGTA and MgCl2 were added during the assay. An advantage of this assay is that it is possible, using microplates, multipipettes and a spectrophotometer coupled to a computer, to semi-automate the procedure.

Animals↗
PubMed1992

Determination of optimal in vitro conditions for caprine alternative complement pathway assay.

Optimal in vitro testing conditions for caprine alternative complement pathway assay were determined. Effects of the following variables were tested: heterologous erythrocytes; pH, ionic strength and Mg2+ ion concentration of the complement diluent; incubation time and temperature. Rabbit erythrocytes were the optimal target cells. The optimal buffer conditions were: pH 8.0, ionic strength 0.06 mmol NaCl and 5mmol Mg2+ ion. Optimal incubation time and temperature were 75 min and 30 degrees C, respectively.

Animals↗
PubMed1978

Regulation by membrane sialic acid of beta1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway.

Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO(4)). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and beta1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by beta1H. A 1% decrease in intact sialic acid was associated with a 1% decrease in beta1H activity in decay-dissociation of membrane bound C3b,Bb. Because removal of the C8 and C9 carbon atoms from the polyhydroxylated side chain of sialic acid by oxidation with NaIO(4) was functionally equivalent to removal of the entire sialic acid moiety, secondary effects of the latter reaction, such as diminution of the negative charge of the membrane or exposure of penultimate galactose residues, were not considered to be responsible for the altered activity of beta1H. These studies suggest that facilitation, by membrane sialic acid residues, of the interaction between bound C3b and beta1H is essential to prevent the particle from effectively activating the alternative pathway.

Animals↗
PubMed1976

Nonspecific complement activation by streptococcal structures. II. Properdin-independent initiation of the alternate pathway.

Complement consumption by isolated membranes and walls from Group A streptococci and various other gram-positive microbes has been tested. These microbial structures were found to activate the alternate complement pathway. However, unlike endotoxin, inulin, or other plant polysaccharides, activation of complement by our material was found to bypass properdin. The activating factor(s) also differs from cobra venom in its/their requirement for factor D. Preliminary experiments suggest this factor isolated from membranes to be a protein and to have a mol wt greater than 40-60,000 daltons. Our studies have led us to speculate that the phylogenetic role of the alternate complement pathway may be the primordial nonspecific defense system which has retained certain fundamental aspects up to the present time.

Antigens, Bacterial↗
PubMed1979

The activation of the alternative complement pathway by fetal calf serum and mouse thymocytes.

Mouse thymocytes activated the alternative complement pathway of mouse serum in the presence of heated fetal calf serum. The activation required C3 from the fetal calf serum but was independent of antibody either in the murine or bovine serum. No other murine cells tested, including erythrocytes, peripheral blood lymphocytes, lymph node cells, spleen cells, and various cultured cell lines, activated the alternative complement pathway as effectively as thymocytes. In addition, sera from species other than cows could not substitute for fetal calf serum. The C3 deposited on thymocytes was in the form of both C3b (immune adherence positive) and C3bi (conglutinable). We propose that the basis of activation in this system is the specific protection of bovine C3b on mouse thymocyte surface.

Absorption↗
PubMed1978

Activation of the alternate complement pathway by peptidoglycan from streptococcal cell wall.

Activation of the alternate complement pathway in human serum by several bacterial components was compared. Peptidoglycan from group A streptococcal cell walls was the most active material, on a weight basis, followed by cell walls, protoplast membranes, and whole cells. The group-specific carbohydrate was inactive. Treatment of peptidoglycan with low concentrations of lysozyme or short periods of sonic treatment enhanced complement activation. High concentrations of lysozyme or extended sonic treatment of peptidoglycan destroyed or greatly reduced the capacity to activate complement. Lysozyme treatment of group A streptococcal cell walls or lipopolysaccharide had no measurable effect. Activation of the alternate complement pathway by group D streptococcal cell walls was destroyed by lysozyme. Activity of peptidoglycan was not inhibited by N-acetyl glucosamine, N-acetyl muramic acid, or D-alanine-D-alanine. Conversion of C3 and factored B by peptidoglycan was shown to occur by immunoelectrophoresis and crossed immunoelectrophoresis.

Animals↗
PubMed1979

Chemotaxigenesis and activation of the alternative complement pathway by encapsulated and non-encapsulated Cryptococcus neoformans.

In the presence of serum, whole cells of encapsulated and non-encapsulated Cryptococcus neoformans generated a chemotactic response by neutrophils. Heat inactivation of serum ablated all chemotactic activity. Cryptococcal polysaccharide was not chemotaxigenic. Assays for alternative complement pathway activation such as depletion of alternative complement pathway factor B or electrophoretic conversion of factor B closely paralleled chemotaxis assays. Cells of encapsulated and non-encapsulated C. neoformans activated the alternative complement pathway, whereas cryptococcal polysaccharide was inactive. Failure of the capsular material to activate the alternative pathway was not due to serotype specificity because polysaccharide of several serotypes failed to achieve activation. The results suggest that chemotaxigenesis and alternative complement pathway activation are functions of the yeast cell wall. The results support our proposal that the cryptococcal capsul does not prevent potential opsonins from reaching binding and activation sites at the yeast cell wall or the release of biologically active soluble cleavage products into the surrounding medium; however, cell wall-bound cleavage products remain bound to the cell wall beneath the capsule. Therefore, they are unable to participate as opsonins in phagocytosis.

Animals↗